Solid phytase compositions

ABSTRACT

The present invention relates to solid enzyme, in particular phytase, compositions stabilized with a lactic acid source such as Corn Steep Liquor (CSL), and methods of producing the same. Preferred compositions additionally comprise starch and disaccharides such as lactose or trehalose.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of Ser. No. 09/410,503 filedOct. 1, 1999 which claims priority or the benefit under 35 U.S.C. 119 ofDanish application no. PA 1998 01251 filed Oct. 2, 1998 and U.S.provisional application No. 60/103,522 filed Oct. 8, 1998, the contentsof which are fully incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to solid phytase compositions whichhave been stabilized with a lactic acid source such as Corn Steep Liquor(CSL), and methods of producing the same.

[0004] 2. Description of Related Art

[0005] The addition of phytase to animal feed to eliminate theanti-nutritional effects of phytic acid is well described, see e.g. WO98/28408 and WO 98/28409.

[0006] The stabilization of liquid phytase formulations with urea,glycerol or sorbitol is disclosed in WO 93/16175.

[0007] Salt-stabilized solid phytase compositions are disclosed in EP 0758 018 A1.

[0008] Plant seeds, cereal grains and legumes are usual components ofanimal feed. Some of those seeds contain phytic acid, and often alsoendogenous phytase enzymes.

[0009] According to investigations performed by the applicant,endogenous phytase activity in animal feed is at a very low level ofaround 0.5 units/g.

[0010] According to e.g. the two above first-cited WO-references, whensupplementary phytase has been added to feed, the phytase activity inthe feed is in the range of 0.01-20 units/g.

SUMMARY OF THE INVENTION

[0011] The present invention relates to solid phytase compositions whichcomprise (a) an enzyme having phytase activity; and (b) a lactic acidsource, wherein the phytase activity of the composition is above 20units/g.

DETAILED DESCRIPTION OF THE INVENTION

[0012] In the present context, the expression “enzyme (or polypeptide)having phytase activity” or “phytase” includes any enzyme capable ofeffecting the liberation of inorganic phosphate from phytic acid or fromany salt thereof (phytates).

[0013] Phytic acid is myo-inositol 1,2,3,4,5,6-hexakis dihydrogenphosphate (or for short myo-inositol hexakisphosphate). In what follows,unless otherwise indicated, the terms “phytic acid” and “phytate,” areused synonymously or at random.

[0014] In the present context, the term “units” means units of enzyme,in particular phytase, activity. Any method for determining phytaseactivity can be used.

[0015] In a preferred embodiment, one unit of phytase activity isdefined as the amount of enzyme that liberates 1 micro mole inorganicortho-phosphate per min. under the following conditions: A pH which iswithin the range of +/−1 pH unit from the optimum pH of the actualenzyme; a temperature which is within the range of +/−20° C. from theoptimum temperature of the actual enzyme; using as a substrate phyticacid or any salt thereof in a suitable concentration.

[0016] Preferably, the substrate is dodeca-sodium phytate in aconcentration of 0.005 mole/l.

[0017] Preferably, the pH is within the range of +/−0.5 pH unit from theoptimum pH; more preferably the pH is the optimum pH.

[0018] Preferably, the temperature is within the range of +/−10° C. fromthe optimum temperature; more preferably the temperature is the optimumtemperature.

[0019] Preferably, the optimum pH and optimum temperature refers to theuse of sodium phytate as a substrate.

[0020] In another preferred embodiment, the phytase activity isdetermined in the unit of FYT, one FYT being the amount of enzyme thatliberates 1 micro mole inorganic ortho-phosphate per min. under thefollowing conditions: pH 5.5; temperature 37° C.; substrate: sodiumphytate (C₆H₆O₂₄P₆Na₁₂) in a concentration of 0.0050 mole/l.

[0021] In a further preferred embodiment, the phytase activity ismeasured using the FTU assay.

[0022] The FYT- and FTU-assays are described in more detail in theexperimental part.

[0023] In preferred embodiments, the phytase activity of the solidcomposition of the invention is above 25, 50, 100, 250, 500, 750 or evenabove 1000 units/g.

[0024] Optionally, the phytase activity of the solid composition isbelow 100,000 units/g, more preferably below 75,000 units/g, even morepreferably below 50,000 units/g, or below 40,000 units/g, or below25,000 units/g, or even below 10,000 units/g, mostly preferred below5,000 units/g.

[0025] Preferred ranges of phytase activity are 25-100,000,25-75,000,35-50,000, or 50-40,000 units/g; more preferably 100-25.000 units/g;even more preferably 500-10.000 units/g; mostly preferred 1000-5000units/g.

[0026] In the present context, any enzyme having phytase activity can beused.

[0027] Phytases have been derived from plants as well as frommicroorganisms. Amongst the microorganisms, phytase producing bacteriaas well as phytase producing fungi are known. From the plant kingdom,e.g. a wheat-bran phytase is known (Thomlinson et al, Biochemistry, 1(1962), 166-171). An alkaline phytase from lilly pollen has beendescribed by Barrientos et al, Plant. Physiol., 106 (1994), 1489-1495.

[0028] Amongst the bacteria, phytases have been described which arederived from Bacillus subtilis (Paver and Jagannathan, 1982, Journal ofBacteriology 151:1102-1108) and Pseudomonas (Cosgrove, 1970, AustralianJournal of Biological Sciences 23:1207-1220). Still further, a phytasefrom E. coli has been purified and characterized by Greiner et al, Arch.Biochem. Biophys., 303, 107-113, 1993).

[0029] Phytase producing yeasts are also described, such asSaccharomyces cerevisiae (Nayini et al, 1984, Lebensmittel Wissenschaftund Technologie 17:24-26. However, this enzyme is probably amyo-inositol monophosphatase (Wodzinski et al, Adv. Appl. Microbiol.,42, 263-303). AU-A-24840/95 describes the cloning and expression of aphytase of the yeast Schwanniomyces occidentalis.

[0030] There are several descriptions of phytase producing filamentousfungi, primarily belonging to the fungal phylum of Ascomycota(ascomycetes). In particular, there are several references to phytaseproducing ascomycetes of the Aspergillus genus such as Aspergillusterreus (Yamada et al., 1986, Agric. Biol. Chem. 322:1275-1282). Also,the cloning and expression of the phytase gene from Aspergillus nigervar. awamori has been described (Piddington et al., 1993, Gene133:55-62). EP 0 420 358 describes the cloning and expression of aphytase of Aspergillus ficuum (niger). EP 0 684 313 describes thecloning and expression of phytases of the ascomycetes Myceliophthorathermophila and Aspergillus terreus.

[0031] Phytases derived from fungi of the phylum Basidiomycota aredisclosed in WO 98/28409 and WO 98/28408.

[0032] Modified phytases or phytase variants are obtainable by methodsknown in the art, in particular by the methods disclosed in EP 0897010,EP 0897985, PCT/DK99/00153 and PCT/DK99/00154. The phytases disclosed ineither of these four patent applications can also be used in thecompositions of the present invention.

[0033] A solid or dry composition is a particulate material comprising,preferably consisting essentially of, or consisting of, freely flowingparticles of a size ranging from (μm) 0.01, or from 1.0, or preferablyfrom around 1 to 1000, or to 1200, or to 1500, or even up to 2000 (μm).

[0034] Preferably, a solid or dry phytase composition is suchcomposition which can be prepared from liquid phytase concentrates e.g.by spray drying, spray cooling (prilling), or any type of granulation.

[0035] For spray drying, no further components need to be added to theliquid phytase concentrate.

[0036] For spray cooling, a meltable component—such as palm oil (and/oranother meltable vegetable oil or fat), hydrogenated palm oil (and/oranother hydrogenated vegetable oil), tallow, hydrogenated tallow or awax functions as a matrix. The phytase and other ingredients, if any,are introduced into the melted, meltable component, and the melt is thenallowed to solidify under particle-forming conditions, typically in aspray drying tower.

[0037] For many uses, however, including the use in animal feed,granulates are usually preferred for a number of reasons. One reason isthat they may readily be mixed with feed components, or more preferably,form a component of a pre-mix which contains other desired feedadditives such as vitamins and minerals.

[0038] The particle size of the enzyme granulates preferably iscompatible with that of the other components of the mixture. Thisprovides a safe and convenient mean of incorporating enzymes into, e.g.,animal feed.

[0039] The size of a particle may be regarded as the greatest lineardimension of the particle; thus, in the case of, e.g., a substantiallyspherical particle (such as a substantially spherical granulateparticle), the particle size in question will be the diameter of theparticle.

[0040] Agglomeration granulates and agglomerated powders may be preparedusing agglomeration technique in a high shear mixer (e.g. Lödige) duringwhich one or more filler materials and the enzyme are co-agglomerated toform granules.

[0041] Absorption granulates are prepared by having cores of one or morecarrier materials to absorb/be coated by the enzyme.

[0042] Typical filler materials are salts such as di-sodium sulphate andcalcium-lignosulphonate. Other fillers are silica, gypsum, kaolin, talc,magnesium aluminium silicate and cellulose fibres. Optionally, binderssuch as dextrins are also included in agglomeration granulates.

[0043] Typical carrier materials may consist of particulate cores havinga suitable particle size. The carrier may be water soluble or waterinsoluble, e.g. starch, e.g. in the form of cassaya or wheat; or a sugar(such as sucrose or lactose), or a salt (such as sodium chloride orsodium sulphate).

[0044] Optionally, the granulates are coated with a coating mixture.Such mixture comprises coating agents, preferably hydrophobic coatingagents, such as hydrogenated palm oil and beef tallow, and if desiredother additives, such as calcium carbonate or kaolin.

[0045] WO 97/39116 discloses preferred processes for making solidcompositions of the invention in the form of enzyme-containing granulesor an enzyme-containing granulate, see in particular the sections of thedetailed description therein headed cores, binders, fillers,plasticizers, fibrous materials, superabsorbents, coating layers,enzymes, other adjunct ingredients (these sections being herebyincorporated by reference herein). However, WO 97/39116 does notdisclose the inclusion in the solid composition of a lactic acid source.

[0046] Preferred methods of preparing phytase granulates are referred toin Example 3.

[0047] Preferred solid compositions of the invention are enzymecompositions. The preferred compositions are concentrated, viz. of anactivity of above 20 units/g. Thus, the concept of solid enzymecomposition comprises in particular, but are not limited to, spray-driedenzyme preparations, enzyme granulates, e.g. agglomeration granulatesand absorption granulates, coated as well as un-coated, andenzyme-containing pre-mixes for animal feed. Phytase is a preferredenzyme.

[0048] Liquid enzyme (phytase) concentrates can e.g. be prepared asfollows: The enzyme source, typically a phytase-containing fermentationbroth, is subjected to a primary separation step (e.g. using a decanter,a centrifuge, or a filter press), followed by a second polish filtrationand/or germ filtration step. Finally the liquid is concentrated, e.g.using ultra filtration, followed by a germ filtration. A typical drymatter content is in the range of 10-30%, preferably 15-25%, morepreferably 17-22%.

[0049] In the present context, “a” generally means “one or more” or “atleast one.” This applies i.a. for the following mandatory or optionalcomponents of the compositions of the invention: Phytase, lactic acidsource, CSL, starch, disaccharide, filler, carrier.

[0050] Unless otherwise indicated, all percentage indications areweight/weight, by reference to dry matter content. Preferably, “units/g”also refers to dry matter content. Dry matter content can be determinedby any method known in the art, such as refractometer or drying in anoven to release humidity.

[0051] Unless otherwise indicated, the expression “above” generallymeans “>”, whereas the expressions “up to” or “below” mean “≦”.

[0052] In the present context a “lactic acid source” or a “lactic acidpreparation” is any composition which comprises the compound lactic acidor any lactates, i.e. any salts thereof (lactic acid is 2-hydroxypropanic acid). Likewise, “lactic acid” as used herein includes anylactates. These expressions are used interchangeably for the lactic acidsource, resp. the lactic acid, as is, and for the dry matter partthereof.

[0053] A non-limiting list of lactic acid sources is the following:Lactic acid and lactates as relatively pure chemical compounds (purityof, say, above 70%, 80%, 90%); lactic acid and lactates as more impuresubstances (purity of, say, above 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%); any natural or synthetical composition which comprises lactic acidin an amount of above 5%, preferably above 10%, 15%, 20%, 25%, 30%, 40%,50%, 70%, 80%, 90%.

[0054] The solid enzyme compositions of the invention preferablycomprise up to 20, preferably up to 15, more preferably up to 10, stillmore preferably up to 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75 or 0.5% lacticacid. The content of lactic acid is preferably above 0.001, preferablyabove 0.002, 0.004, 0.006, 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1,0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, or above 0.3%.Preferred ranges of content of lactic, acid are 0.01-10%, 0.02-9%,0.03-8%, 0.04-7%, 0.05-6%, 0.06-7%, 0.07-6%, 0.08-5%, 0.09-4%, or0.1-3%.

[0055] Any assay for lactic acid can be used. Preferred lactic acidassays are from SIGMA: (1) Assay kit catalogue no. 735-10 (enzymaticalassay, lactate degraded to pyruvate and hydrogen peroxide in thepresence of oxidase); or (2) Assay kit catalogue no. 826-A and 826-B(ultraviolet, endpoint, lactate converted into pyruvic acid in thepresence of lactate dehydrogenase and NAD).

[0056] A preferred lactic acid source is Corn Steep Liquor or CSL. CSLis a commercially available product, see for instance Merck Index, 1996,4^(th) edition, Index no. 2598. It is a viscous yellowish or dense brownliquid obtained by concentration of corn steep water. The dry mattercontent is usually 45-55%, preferably 48-52%. The pH is in the range of3-5, preferably 3.5-4.5. The protein content (Dry Matter) is typically30-50%, preferably 35-45%. The acidity (as lactic acid) is typically10-30% (Dry Matter), preferably 12-25%.

[0057] “CSL” as used herein refers to the product as such, or to its drymatter part.

[0058] In a preferred embodiment, the solid composition of the inventioncomprises 0.01-15%, preferably 0.1-10%, more preferably 1-5% of CSL.

[0059] For analyzing CSL-content, any method can be used. A preferredHPLC method for fingerprinting and quantifying CSL is indicated inExample 8. Another preferred method is Head Space Gas Chromatograph(HS-GC), preferably combined with mass spectrometry (MS).

[0060] In a preferred embodiment, the solid composition of the inventionadditionally comprises a starch source, typically in an amount of0.1-20%, more preferably 0.2-10%, still more preferably 1-5%.

[0061] The concept of a starch source includes any natural or syntheticpolysaccharides comprising glucose units interconnected by alpha-1,4- oralpha-1,6-linkages. Purity is preferably above 10, 20, 30, 40, 50, 60,70, 80, 90, or 100%. A preferred starch source is Wheat Starch, which isa commercially available product. The expression “starch source”includes the starches and modified starches described in the sectionheaded “Cores” of WO 97/39116, cited above.

[0062] In another preferred embodiment, the solid composition of theinvention additionally comprises a disaccharide, preferably in an amountof 0.01-15%, even more preferred 0.1-10%, even more preferrably 1-5%.

[0063] The concept of disaccharides includes any natural or syntheticdisaccharides, whatever the monomers, and whatever the linkage type.Examples of such disacharides are maltose, lactose, cellobiose, sucrose,trehalose (non-limiting list). Preferably, the disaccharides are of apurity of above 10, 20, 30, 40, 50, 60, 70, 80 or even 90%. Preferreddisaccharides are lactose and trehalose (alpha-D-glucosealpha-D-glucopyranoside, alpha-1,1 linkage).

[0064] In the process of the invention, all steps, e.g. those indicatedin claim 14, can be performed simultaneously or sequentially. E.g. steps(i) and (ii) sequentially or preferably simultaneously (mixing thelactic acid source and the phytase before spraying it onto the carrier);steps (iii) and (iv) simultaneously or sequentially, preferablysimultaneously, in the same apparatus; applies also to “together with”of claim 12.

[0065] Further preferred embodiments of the invention are the following:

[0066] A solid composition which comprises at least one enzyme havingphytase activity, and Corn Steep Liquor (CSL), wherein the phytaseactivity of the composition is in the range of 20-50.000 units/g. Apreferred amount of CSL is within the range of 0.01-15% (dry mattercontent and w/w). Preferably, the composition additionally comprisesWheat Starch (WS), preferably in an amount within the range of 0.01-20%(dry matter content and w/w);

[0067] A process for preparing a granulate composition having a phytaseactivity in the range of 20-50.000 units/g, which method comprises thesteps of (i) spraying a liquid phytase concentrate onto a carrier; (ii)spraying CSL onto the carrier; (iii) mixing; and (iv) drying;

[0068] A process for preparing a spray dried solid composition having aphytase activity in the range of 20-50.000 units/g, which methodcomprises the step of adding CSL to a liquid phytase concentrate beforespray drying it.

[0069] The activities of the solid phytase compositions prepared in thepresent examples are in the range of 1000-3000 FTU/g before storage.

EXAMPLE 1

[0070] FYT Assay

[0071] 10 μl diluted enzyme samples (diluted in 0.1 M sodium acetate,0.01% Tween 20, pH 5.5) are added into 250 μl 5 mM sodium phytate(Sigma) in 0.1 M sodium acetate, 0.01% Tween 20, pH 5.5 (pH adjustedafter dissolving the sodium phytate; the substrate is preheated) andincubated for 30 minutes at 37° C. The reaction is stopped by adding 250μl 10% TCA and free phosphate is measured by adding 500 μl 7.3 g FeSO4in 100 ml molybdate reagent (2.5 g (NH₄)₆Mo₇O_(240.4)H₂O in 8 ml H₂SO₄diluted to 250 ml). The absorbance at 750 nm is measured on 200 Alsamples in 96 well microtiter plates. Substrate and enzyme blanks areincluded. A phosphate standard curve is also included (0-2 mMphosphate). 1 FYT equals the amount of enzyme that releases 1 μmolphosphate/min at the given conditions.

[0072] FTU Assay

[0073] One FTU is defined as the amount of enzyme, which at standardconditions (37° C., pH 5.5; reaction time 60 minutes and startconcentration of phytic acid 5 mM) releases phosphate equivalent to 1μmol phosphate per minute.

[0074] 1 FTU=1 FYT

[0075] The FTU assay is preferred for phytase activity measurements onanimal feed premixes and the like complex compositions.

[0076] Reagents/Substrates

[0077] Extraction Buffer for Feed etc.

[0078] This buffer is also used for preparation of PO₄-standards andfurther dilution of premix samples.

[0079] 0.22 M Acetate Buffer with Tween 20 pH 5.5

[0080] 30 g sodium acetate trihydrate (MW=136.08 g/mol) e.g. Merck Art46267 per liter and 0.1 g Tween 20 e.g. Merck Art 22184 pr. liter areweighed out.

[0081] The sodium acetate is dissolved in demineralized water.

[0082] Tween 20 is added, and pH adjusted to 5.50+0.05 with acetic acid.

[0083] Add demineralized water to total volume.

[0084] Extraction Buffer for Premix

[0085] 0.22 M acetate buffer with Tween 20, EDTA, PO₄ ³⁻ og BSA.

[0086] 30 g sodium acetate trihydrate e.g. Merck Art 6267 per liter.

[0087] 0.1, g Tween 20 e.g. Merck Art 22184 per liter.

[0088] 30 g EDTA f.eks. Merck Art 8418 per liter.

[0089] 20 g Na₂HPO₄, 2H₂O e.g. Merck Art 6580 per liter.

[0090] 0.5 g BSA (Bovine Serum Albumine, e.g. Sigma Art A-9647 perliter.

[0091] The ingredients are dissolved in demineralized water, and pH isadjusted to 5.50±0.05 with acetic acid.

[0092] Add demineralized water to total volume.

[0093] BSA is not stable, and must therefore be added the same day thebuffer is used.

[0094] 50 mM PO₄ ³⁻ Stock Solution

[0095] 0.681 g KH₂PO₄ (MW=136.09 g/mol) e.g. Merck Art 4873 is weighedout and dissolved in 100 ml 0.22 M sodium acetate with Tween, pH 5.5.

[0096] Storage stability: 1 week in refrigerator.

[0097] 0.22 M Acetate Buffer pH 5.5 without Tween

[0098] This buffer is used for production of phytic acid substrate).

[0099] 150 g sodium acetate trihydrate (MW=136.08) e.g. Merck Art 6267is weighed out and dissolved in demineralized water, and pH is adjustedwith acetic acid to 5.50±0.05.

[0100] Add demineralized water to 5000 ml.

[0101] Storage stability: 1 week at room temperature.

[0102] Phytic Acid Substrate; 5 mM Phytic Acid

[0103] The volume of phytic acid is calculated with allowance for thewater content of the used batch.

[0104] If the water content is e.g. 8.4% the following is obtained:

(0.005 mol/l×923.8 g/mol)/(1/0.084)=5.04 g/l

[0105] Phytic acid (Na-salt) (MW=923.8 g/mol) e.g. Sigma P-8810 isweighed out and dissolved in 0.22 M acetate buffer (without Tween).Addition of (diluted) acetic acid increases the dissolution speed.

[0106] pH is adjusted to 5.50±0.05 with acetic acid.

[0107] Add 0.22 M acetate buffer to total volume.

[0108] 21.7% Nitric Acid Solution

[0109] For stop solution.

[0110] 1 part concentrated (65%) nitric acid is mixed into 2 partsdemineralized water.

[0111] Molybdate Reagent

[0112] For stop solution.

[0113] 100 g ammonium heptamolybdate tetrahydrate (NH₄)₆Mo₇O₂₄, 4H₂Oe.g. Merck Art 1182 is dissolved in demineralized water. 10 ml 25% NH₃is added.

[0114] Add demineralized water to 1 liter.

[0115] 0.24% Ammonium Vanadate

[0116] Bought from Bie & Berntsen.

[0117] Molybdate/Vanadate Stop Solution

[0118] 1 part vanadate solution (0.24% ammonium vanadate)+1 partmolybdate solution are mixed. 2 parts 21.7% nitric acid solution areadded.

[0119] The solution is prepared not more than 2 hours before use, andthe bottle is wrapped in tin foil.

[0120] Samples

[0121] Frozen samples are defrosted in a refrigerator overnight.

[0122] Sample size for feed samples: At least 70 g, preferably 100 g.

[0123] Feed Samples

[0124] Choose a solution volume which allows addition of buffercorresponding to 10 times the sample weight, e.g. 100 g is dissolved in1000 ml 0.22 M acetate buffer with Tween, see enclosure 1. Round up tonearest solution volume.

[0125] If the sample size is approx. 100 g all the sample is ground in acoffee grinder and subsequently placed in tared beakers. The sampleweight is noted. It is not necessary to grind not-pelleted samples. If asample is too big to handle, it is sample split into parts of approx.100 g.

[0126] Magnets are placed in the beakers and 0.22 M acetate buffer withTween is added.

[0127] The samples are extracted for 90 minutes.

[0128] After extraction the samples rest for 30 minutes to allow for thefeed to sediment. A 5 ml sample is withdrawn with a pipette. The sampleis taken 2-5 cm under the surface of the solution and placed in acentrifuge glass, which is covered by a lid.

[0129] The samples are centrifuged for 10 minutes at 4000 rpm.

[0130] Premix Samples

[0131] Choose a solution volume which allows addition of buffercorresponding to 10 times the sample weight. Round up to nearestsolution volume.

[0132] If the samples have been weighed (50-100 g) all of the sample isplaced in tared beakers. The sample weight is noted. If a sample is toobig to handle, it is split into parts of approx. 100 g.

[0133] Magnets are placed in the beakers and 0.22 M acetate buffer withTween, EDTA and P₄ ³⁻ is added.

[0134] The samples are extracted for 60 minutes.

[0135] After extraction the samples rest for 30 minutes to allow for thepremix to sediment. A 5 ml sample is withdrawn with a pipette. Thesample is taken 2-5 cm under the surface of the solution and placed in acentrifuge glass, which is covered by a lid.

[0136] The samples are centrifuged for 10 minutes at 4000 rpm.

[0137] Analysis

[0138] Extracts of feed samples are analyzed directly.

[0139] Extracts of premix are diluted to approx. 1.5 FTU/g (A₄₁₅ (mainsample)<1.0).

[0140] 0.22 M acetate buffer with Tween 20 is used for the dilution.

[0141] Main Samples

[0142] 2×100 ml of the supernatant from the extracted and centrifugedsamples are placed in marked glass test tubes and a magnet is placed ineach tube.

[0143] When all samples are ready they are placed on a water bath withstirring. Temperature: 37° C.

[0144] 3.0 ml substrate is added.

[0145] Incubation for exactly 60 minutes after addition of substrate.

[0146] The samples are taken off the water bath and 2.0 ml stop solutionis added (exactly 60 minutes after addition of substrate).

[0147] The samples are stirred for 1 minute or longer.

[0148] Feed samples are centrifuged for 10 minutes at 4000 rpm (It isnot necessary to centrifuge premix samples).

[0149] Blind Samples

[0150] 100 ml of the supernatant from the extracted and centrifugedsamples are placed in marked glass test tubes, and a magnet is placed ineach tube.

[0151] 2.0 ml stop solution is added to the samples.

[0152] 3.0 ml substrate is added to the samples.

[0153] The samples are incubated for 60 minutes at room temperature.

[0154] The feed samples are centrifuged for 10 minutes at 4000 rpm (itis not necessary to centrifuge premix samples).

[0155] Standards

[0156] 2×100 ml are taken from each of the 8 standards and also 4×100 ml0.22 M acetate buffer (reagent blind).

[0157] A₄₁₅ is measured on all samples.

[0158] Calculation

[0159] FTU/g=μmol PO₄ ³⁻/(min*g (sample))

[0160] C g sample is weighed out (after grinding).

[0161] 100 μl is taken from the extracted and centrifuged sample.

[0162] PO₄ ³⁻ standard curve is linear.

[0163] From the regression curve for the PO₄ ³⁻ standard the actualconcentration of the sample is found (concentration in mM):

[0164] [PO₄ ³⁻]=(x−b)/a x=A₄₁₅ a=slope b=intercept with y-axis

[0165] μmol PO₄ ³⁻/min={[PO₄ ³⁻] (mM)×Vol (liter)×1000 μmol/mmol}/t

[0166] t=incubation time in minutes.

[0167] Vol=sample volume in liter=0.0001 liter

[0168] 1000=conversion factor from mmol to μmol

[0169] FTU/g_(prøve)={(x−b)×Vol×1000×F_(p)}/{a×t×C}

[0170] C=gram sample weighed out

[0171] F_(p)=Relation between the sample taken out and the total sample(after extraction).

[0172] Example: 0.100 ml taken from 1000 ml→F_(p)=1000/0.100=10000.

[0173] Reduced expression with insertion of the following values:

[0174] t=60

[0175] Vol=0.0001 l

[0176] F=10000

[0177] FTU/g_(prøve)={(x−b)×0.0001×1000×10000}/{a×60×C}

EXAMPLE 2

[0178] Preparation of a Concentrated Liquid Phytase Preparation

[0179] The phytase derived from Peniophora lycli is expressed inAspergillus oryzae, fermented and purified, essentially as described inWO 98/28408. The resulting liquid phytase concentrate is a UF (ultrafiltration) concentrate of a dry matter content of 18%. pH is adjustedto 5.

EXAMPLE 3

[0180] Preparation of Phytase Granulates

[0181] A coated phytase granulate with 1.5% CSL is prepared as follows:

[0182] 14.68 kg of a powder composition with the formulation

[0183] 0.75 kg kaolin, Speswhite, English China Clay

[0184] 1.80 kg of fibrous cellulose, Arbocel BC 200

[0185] 11.23 kg finely ground sodium sulphate

[0186] 0.90 kg Carbohydrate binder, Tackidex G155 from Roquette

[0187] is mixed in a Lödige mixer FM 50 and sprayed with 3.15 kg of aspraying liquid consisting of 1.68 kg of water, 0.625 kg of Corn SteepLiquor (Concentrated Corn Steep Liquor (CCSL) supplied by Amylum N.V.with a dry matter content of 48%) and 0.84 kg of Phytase concentrate(18% dry matter content) prepared as described in Example 2. During andafter spraying the moist mixture is exposed to a compacting andgranulation influence from the multiple set of knives, as described inExample 1 of U.S. Pat. No. 4,106,991.

[0188] The percentage of CSL in this as yet un-coated raw granulate iscalculated as follows:0.625×0.48/(14.68+0.625×0.48+0.84×0.18)=0.300/(14.68+0.30+0.672)=0.300/15.652=1.917%−2%.

[0189] The granulate is dried in a fluid bed to a water content below3%, resulting in a light coloured granulate with the following particledistribution:

[0190] 10.5%>1100 μm (micro meter)

[0191] 92.0%>300 μm

[0192] 8.0%<300 μm

[0193] The granulate is finally sifted to get a product with theparticle range 300 μm to 1100 μm, and 6 kg of granulate is coated at 80°C. with 9% fully hydrogenated palm oil, followed by 22.5% of kaolin,Speswhite (dry matter content in 100 g coating material: 22.5 g+9 g=31.5g), in a manner as described in U.S. Pat. No. 4,106,991, Example 22.

[0194] The content of CSL in the resulting final product, the coatedgranulate, is reduced as compared to the CSL content of the rawgranulate as follows: 1.917%/1.315=1.458%−1.5%.

[0195] The granulate is sifted to obtain a product with the particlerange 300 μm to 1200 μm.

[0196] The control granulate used below is prepared as described above,except for no CSL being added.

[0197] Granulates additionally comprising wheat starch and lactose ortrehalose are prepared in a corresponding manner.

EXAMPLE 4

[0198] Storage Stability of Phytase Granulates in Premix

[0199] The phytase granulates indicated in Table 1 below are preparedaccording to Example 3. “Control” indicates a phytase granulate preparedaccording to the method of Example 3, but with no addition of CSL.

[0200] The granulates are weighed directly into each vial. The exactweight of the granulate is recorded. The vials are covered with a cleantowel and left at room temperature overnight.

[0201] The premix ENGA 1-02/Nordkorn. Product. No: 015384 Artikel Nr.8259 (25 kg drums) is mixed in a Lödige mixer to ensure an evendistribution of the premix components and filled into plastic bags with≈3 kg premix in each bag.

[0202] The composition of the premix is as follows (per kilo): 5000000IE Vitamin A 1000000 IE Vitamin D3 13333 mg Vitamin E 1000 mg Vitamin K3750 mg Vitamin B1 2500 mg Vitamin B2 1500 mg Vitamin B6 7666 mcg VitaminB12 12333 mg Niacin 33333 mcg Biotin 300 mg Folic Acid 3000 mgCa-D-Panthothenate 1666 mg Cu 16666 mg Fe 16666 mg Zn 23333 mg Mn 133 mgCo 66 mg I 66 mg Se 5.8 % Calcium 25 % Sodium

[0203] 50 g±1 g of premix is added to each vial and the vials are closedwith a screw-on lid. The premix is added using an adjustable cylindrical“scoop” adjusted to give a volume corresponding to 50 g. The vials aremixed by hand until the granulates are evenly distributed in the premix.

[0204] The 0 week samples (closed vials), defining for each granulatethe level of 100% activity, are frozen immediately after completion ofthe sample preparation. The samples which are to be stored at 30° C. arere-opened. The open vials are placed in plastic boxes containing 1 litreof glycerol adjusted with water to 43% rH (62% refractometer dry mattermeasured on a sugar scale) corresponding to ≈10% water in the samples.The lids of the plastic boxes are sealed with strong tape. This meansthat the water activity is 0.43 during the whole test period of 13weeks.

[0205] After completion of the storage period the samples are removedfrom the glycerol boxes, closed with screw on lids and frozen.

[0206] The samples are defrosted in a refrigerator (5° C.) overnightprior to analysis.

[0207] The 0 week samples stored at −18° C. and the correspondingsamples stored at 30° C. are analysed the same day in order to eliminateday-to-day and person-to-person variation.

[0208] The results are shown in Table 1 below; CSL=Corn Steep Liquor andWS=Wheat Starch.

[0209] Phytase granulates containing 2% CSL, 3% CSL, and 2% CSL plus 5%WS showed similar performance. TABLE 1 Percentage residual activityfollowing 13 Granulate code Granulate weeks storage at 30° C. 1 Controlgranulate 61% 2 Control granulate 64% 3 1.5% CSL 81% 4 1.5% CSL 86% 51.5% CSL 84% 6 1.5% CSL 85% 7 1.5% CSL + 3.8% WS 84% 8 1.5% CSL + 3.8%WS 90% 9 1.5% CSL + 3.8% WS 84%

EXAMPLE 5

[0210] Storage Stability of Phytase Granulates in Feed

[0211] The phytase granulates indicated in Table 2 below are preparedaccording to Example 3. “Control” indicates a phytase granulate preparedaccording to the usual standard method of Example 3, except for neitherWS nor CSL nor disaccharides being added.

[0212] The samples of granulates in feed are prepared at BioteknologiskInstitut, Kolding, Denmark.

[0213] The composition of the feed is as follows:

[0214] 74.0% wheat

[0215] 20.7% roasted soy cake

[0216] 5.0% soy oil

[0217] 0.3% Premix Enga 1-02/Nordkorn

[0218] The feed is dried to a water content of ≦10% water beforeaddition of the phytase granulates.

[0219] The granulate batches are mixed into feed and the mixture ispelletized at 65° C.

[0220] The feed pellets are sample split and filled into 100 ml samplevials.

[0221] The 0 week samples, defining for each granulate the level of 100%activity, are closed with screw on lids and kept at −18° C.

[0222] The samples which are to be stored at 30° C. are not closed. Theopen vials are placed in plastic boxes containing 1 litre of glyceroladjusted with water to 43% rH (62% refractometer dry matter measured ona sugar scale) corresponding to # 10% water in the samples. The lids ofthe plastic boxes are sealed with strong tape. This means that the wateractivity is 0.43 during the whole test period of 13 weeks.

[0223] After completion of the storage period the samples are removedfrom the glycerol boxes, closed with screw on lids and frozen. Thesamples are defrosted in a refrigerator (5° C.) overnight prior toanalysis.

[0224] Samples for the homogeneity test are kept refrigerated at +5° C.until analysis.

[0225] The mash feed, the feed heated to 65° C., and the feed pelletswithout added enzyme all contained ≈0.5 FTU/g feed as expected.

[0226] 5 samples of the mash feed with enzyme added and heated to 65° C.are analysed for homogeneity. The relative standard deviation is 2% to11%. In conclusion, the homogeneity is acceptable.

[0227] 5 samples of the feed pellets are analysed for homogeneity. Therelative standard deviation is 2% to 10%. In conclusion, the homogeneityis acceptable.

[0228] The storage stability is measured after 13 weeks. The 0 weeksamples stored at −18° C. and the corresponding samples stored at 30° C.are analysed the same day in order to eliminate day-to-day andperson-to-person variation.

[0229] The results of the phytase residual activity measurements areshown in Table 2 below (endogenous activity has been subtracted from thetotal activity before calculating the residual activity); CSL=Corn SteepLiquor and WS=Wheat Starch.

[0230] Phytase granulates containing 2% CSL, 3% CSL, and 2% CSL plus 5%WS showed similar performance. TABLE 2 Percentage residual activityfollowing 13 Granulate code Granulate weeks storage at 30° C. 1 Controlgranulate 53% 2 Control granulate 55% 3 1.5% CSL 74% 4 1.5% CSL 89% 51.5% CSL 82% 6 1.5% CSL 73% 7 1.5% CSL + 3.8% WS 82% 8 1.5% CSL + 3.8%WS 91% 9 1.5% CSL + 3.8% WS 89%

EXAMPLE 6

[0231] Phytase Granulates per se; Granulation Yield and StorageStability

[0232] The liquid concentrate of Example 2 was used to prepareexperimental solid phytase compositions according to the method ofExample 3.

[0233] In a first granulation experiment, the disaccharide lactose wasadded in an amount of 2% together with 3% of the lactic acid source CornSteep Liquor (CSL).

[0234] In a second granulation experiment, 3% Wheat Starch (WS) wasapplied—in addition to the two components of the first experiment.

[0235] In a third granulation experiment, the disaccharide trehalose, inan amount of 2%, as well as 3% WS, was added together with 3% of thelactic acid source CSL.

[0236] The effect on granulation yield and storage stability of theresulting granulates per se is examined.

[0237] Granulation yield is calculated as phytase units remaining in theproduct leaving the granulation unit, relative to phytase units of theliquid concentrate entering the unit.

[0238] The storage stability of the resulting phytase granulatecomposition per se is examined using the following rather strictconditions: 4 weeks, 40° C. and a relative humidity of 60%.

[0239] The results are shown in Table 3 below. TABLE 3 Granulation yieldStorage stability Experiment Batch 22 Batch 24 Batch 22 Batch 24 Control75% 70% 43% 47% CSL + lactose 80% 67% 47% 50% CSL + WS + lactose 81% 79%56% 54% CSL + WS + trehalose 85% 82% 63% 62%

EXAMPLE 7

[0240] Storage Stability of a Granulate Composition of Another Phytase

[0241] A liquid phytase concentrate and a solid composition—i.e. agranulate—was prepared according to the teachings of Examples 2 and 3,using a so-called consensus phytase as described in EP 0897010.

[0242] Granulation experiments were conducted essentially as describedin Example 6. However, for storage stability samples are also stored at30° C. The results are shown in Table 4 below. TABLE 4 Storage stabilityGranulation 30° C. 40° C., 60% RH Experiment yield 8 weeks 17 weeks 4weeks Control 82% 85% 85% 40% CSL + WS + 92% 97% 95% 48% lactose

EXAMPLE 8

[0243] Characterization of CSL using High Performance LiquidChromatography (HPLC)

[0244] 15 samples from various batches of CSL from various suppliers(Roquette Freres, 4 Rue Patou, F-59022 Lille Cedex, France; Staral s.a.,Z.I.ET Portuaire, B.P. 32, F-67390 Marckolsheim, France; and CerestarScandinavia A/S, Skovlytoften 33, DK-2840 Holte, Denmark) are tested asdescribed below.

[0245] Carrez-Precipitation

[0246] Weigh 5.0 g CSL into a 100 ml flask. Add 40 ml MQ-water(demineralized water filtered through a Milli-q filter) and incubate at70° C. for 15 minutes while shaking at 200 rpm. Add 12 ml ofCarrez-1-solution (Potassium-hexacyanoferrat(II)-tri hydrate) and shake.Add 12 ml Carrez-II-solution (Zinc sulphate-hepta hydrate) and shake.Add 20 ml 0.5N NaOH and shake. Let cool and add MQ-water ad 100 ml,shake. 10 ml of this preparation is transferred to a vial andcentrifuged for 10 minutes at 4000 rpm. The supernatant is filtered at a0.5 μm filter for HPLC analysis. Each sample is analyzed twice, includesample blinds (MQ-water and Carrez-solutions).

[0247] Chromatography Parameters

[0248] Column: Supelcosil LC-18-DB, No. 088877AE

[0249] Detector: Shimadzu SPDM6A-diodearray from 220 nm to 350 nm.

[0250] Data analysis: For analyzing data, use peak areas resulting fromintegration at 260 nm. Pump: HP 1080 gradient pump Eluents: A) MQ-waterB) 30% MeOH C) 60% MeOH D) 90% MeOH Gradients:  0 min A 15 min A 35 minB 50 min C 60 min C 65 min D 70 min D 75 min C 80 min B 85 min A 90 minA

[0251] The results of variable statistics on 15 samples of CSL analyzedby HPLC are shown in Table 5 below: TABLE 5 Peak Standard retentionDeviation time Min Max Mean (SD) % SD 6791 22154 114227 79192 2597832.80⁷ 7445 1934348 2326644 2135867 110911 5.19¹ 10331 4746 132048 4450352860 118.78 11844 0 3020 201 780 387.30 12440 124723 183826 15594617166 11.01⁴ 14124 79613 186731 119578 35032 29.30⁶ 15322 0 27204 1320511537 87.37 16187 314623 380627 346898 21271 6.13² 18831 18494 148325130989 10481 8.00³ 26373 0 8482 2288 3454 150.99 26833 0 59860 2562219252 75.14 27672 0 46259 24388 13200 54.12 28053 0 11383 3085 3169102.71 28762 5643 60078 31762 20966 66.01 29491 3657 14650 8970 335337.38⁸ 29926 40184 89538 52754 14807 28.07⁵ 30607 0 44749 14732 15523105.37 30951 0 19732 2423 5786 238.80 31825 0 16090 2624 4909 187.0632454 0 15725 1288 4050 314.36 32636 0 28268 7516 9534 126.85 33068 036398 4570 9707 212.42 33394 0 96671 30197 33359 110.47 33646 0 248564042 7600 188.02 34108 0 11826 2144 3922 182.94 34464 0 29248 9794 899091.79 35309 0 14392 8793 6491 73.82 36826 0 29619 9667 10423 107.8242457 0 45570 32023 12404 38.73⁹ 42971 29905 102074 45303 18156 40.08¹⁰43427 0 49318 6293 14306 227.33 43812 0 13837 5851 4924 84.16 45519 012487 3570 4815 134.87 46032 0 11850 4728 4756 100.59 46654 0 3635723766 11561 48.65 47034 0 37769 17540 11212 63.92 47268 0 20271 54146969 128.72 47784 0 5218 1569 2091 133.25 48494 0 5858 1872 1595 85.2048859 0 10935 2923 3748 128.25 49180 0 24091 11016 8573 77.82 49467 023885 10146 8372 82.52 49905 0 22011 4205 8506 202.31

[0252] In the % SD column of Table 5, characteristic peaks are indicatedby way of a superscript number (1,2,3, - - - ,9,10). In what follows,these peaks will be referred to as peak-1, peak-2, peak-3, - - - ,peak-9, peak-10, respectively. The whole group of ten peaks is referredto as peaks 1-10. Sub-groups are referred to by analogy, e.g. peaks 1-5for the five peaks numbered 1 to 5, peaks 1,3,5 for peak-1, peak-3 andpeak-5 etc. Thus, the presence of one or more of these peaks in a sampleis indicative of the presence of CSL. In preferred embodiments, thepresence of one, two, three, four, five, six, seven, eight, nine or allten of these peaks is indicative of the presence of CSL. In morepreferred embodiments, the presence of five, seven, eight or ten peaksis indicative of the presence of CSL. The presence of five peaks is mostpreferred.

[0253] For samples of an unknown content of CSL, suitable dilutions arefound using simple trial-and-error techniques.

[0254] The above qualitative method can be made quantitative bycomparing with a batch denominated by Roquette Freres to be a standardbatch. A particularly preferred standard CSL batch from Roquette Freresis SOLULYS® L 48 L CAS No. 66071-94-1, EINECS: 266-113-4.

1. A solid phytase composition comprising: (a) an enzyme having phytaseactivity, and (b) a lactic acid source, wherein the phytase activity ofthe composition is above 20 units/g.
 2. The composition of claim 1,wherein the lactic acid source comprises at least 10% lactic acid. 3.The composition of claim 1, which comprises lactic acid in an amount of0.01-10%.
 4. The composition of claim 1, wherein the lactic acid sourceis Corn Steep Liquor (CSL).
 5. The composition of claim 4, thechromatogram of which, when analyzed by HPLC according to Example 8herein, reveals the presence of one or more of peaks 1-10.
 6. Thecomposition of claim 4, comprising CSL in an amount of 0.01-15%.
 7. Thecomposition of claim 1, further comprising a starch source.
 8. Thecomposition of claim 1, further comprising a disaccharide.
 9. Thecomposition of claim 1, additionally comprising a carrier material. 10.The composition of claim 1, additionally comprising a filler material.11. The composition of claim 1, additionally comprising one or morevitamins, one or more minerals or a mixture of both.
 12. A process forpreparing a solid phytase composition which comprises drying a lacticacid source together with an enzyme having phytase activity, wherein thephytase activity of the solid composition is above 20 units/g.
 13. Theprocess of claim 12, wherein the lactic acid source is Corn Steep Liquor(CSL).
 14. The process of claim 12, wherein the solid phytasecomposition is a phytase granulate composition, and which processcomprises: (a) spraying the enzyme having phytase activity onto acarrier; (b) spraying the lactic acid source onto the carrier; (c)mixing; and (d) drying.
 15. The process of claim 12, wherein the dryingis a spray-drying.
 16. The process of claim 12, further comprising theaddition of one or more starch sources.
 17. The process of claim 12,further comprising the addition of one or more disaccharides.
 18. Asolid composition having a phytase activity above 20 units/g and beingobtainable by the process of claim
 12. 19. A solid phytase compositionconsisting essentially of: (a) an enzyme having a phytase activity ofabove 20 FYT/g of the composition, and (b) a lactic acid source in anamount of 0.01-15% by weight to provide lactic acid in an amountsufficient to stabilize the enzyme.
 20. The composition of claim 19,wherein the lactic acid is present in an amount of 0.01-10%.
 21. Thecomposition of claim 19, having a chromatogram determined by HPLC, whichhas one or more of peaks 1-10.
 22. The composition of claim 19, furtherconsisting essentially of a starch material.
 23. The composition ofclaim 19, further consisting essentially of a disaccharide.
 24. Thecomposition of claim 19, further consisting essentially of a carriermaterial.
 25. The composition of claim 19, further consistingessentially of a filler material.
 26. The composition of claim 19,further consisting essentially of one or more vitamins, one or moreminerals or a mixture of both.
 27. The solid phytase composition ofclaim 19, wherein the enzyme has a phytase activity of at least 25 FYT/gof the composition.
 28. The solid phytase composition of claim 27,wherein the enzyme has a phytase activity of at least 50 FYT/g of thecomposition.
 29. The solid phytase composition of claim 28, wherein theenzyme has a phytase activity of at least 100 FYT/g of the composition.30. The solid phytase composition of claim 29, wherein the enzyme has aphytase activity of at least 250 FYT/g of the composition.
 31. The solidphytase composition of claim 30, wherein the enzyme has a phytaseactivity of at least 500 FYT/g of the composition.
 32. The solid phytasecomposition of claim 31, wherein the enzyme has a phytase activity of atleast 750 FYT/g of the composition.
 33. The solid phytase composition ofclaim 32, wherein the enzyme has a phytase activity of at least 1000FYT/9 of the composition.